Determinants of linear growth and predictors of severe stunting during infancy in rural Malawi.

UNLABELLED: Stunting is common among children under 5 y of age in sub-Saharan Africa. Several risk factors have been associated with poor growth but few studies have prospectively addressed the development of linear growth faltering and stunting during the first year of life. The present study was designed to analyse typical growth among rural Malawian infants, focusing particularly on the impact of birth size, adherence to feeding guidelines and morbidity in the development of severe stunting during infancy. A community-based cohort of 613 singleton newborns was prospectively followed by monthly home visits. Data were collected on the children's socioeconomic background, maternal size and weight gain during pregnancy, birth events, morbidity, breastfeeding and complementary feeding, growth and mortality. Univariate and multivariate analyses were used to determine associations between predictor variables and poor linear growth. The proportions of stunted infants (Height-for-age Z-score < -2) at 3, 6 and 9 mo of age were 27%, 51%, and 63%, respectively. At I y of age, over two-thirds (71%) of the infants were at least moderately (HAZ < -2) and 31% severely stunted (HAZ < -3). CONCLUSION: The strongest predictor of severe stunting at 12 mo of age was small birth size. Other variables independently associated with this outcome included inappropriate complementary feeding, high morbidity, maternal short stature, male gender, and home delivery. Faltering of linear growth started soon after birth and continued throughout infancy. Interventions increasing birth size could have a significant role in the prevention of early childhood stunting. The ideal strategy should also emphasize the importance of appropriate infant feeding and decreasing the number of illness episodes amongst the infants.

Molecular cloning, sequencing analysis and expression of the catalase-peroxidase gene from Halobacterium salinarum.

The gene encoding catalase-peroxidase was cloned from chromosomal DNA from the Archaea, Halobacterium salinarum. The nucleotide sequence of a 3.5 kb fragment, containing the catalase-peroxidase gene and its flanking regions was determined. A 2.16 kb open reading frame was obtained, encoding the enzyme which was comprised of 720 amino acid residues with a calculated molecular weight of 80 kDa. The deduced amino acid sequence of the H. salinarum catalase-peroxidase showed a high degree of identity to other bifunctional catalase-peroxidases. A transcriptional start site was identified 183 bp upstream of the translational start codon. Southern blot analysis indicated that catalase-peroxidase was a single copy gene. The Archaeal catalase-peroxidase gene was expressed in Escherichia coli, and the expressed fusion protein exhibited both catalase and peroxidase activities.

Breastfeeding and complementary feeding practices in rural Malawi.

UNLABELLED: To facilitate optimal growth of newborns, many countries have developed infant feeding recommendations, usually suggesting 4-6 mo of exclusive breastfeeding and then the gradual introduction of complementary foods. We prospectively studied the changes in infant diets and predictors of adherence to national infant feeding recommendations in a cohort of 720 newborn babies in rural Malawi, Sub-Saharan Africa. Monthly interviews of the main guardians indicated that breastfeeding was universal for 18 mo. As most babies were given water or other supplemental foods soon after birth, the exclusive breastfeeding rates were only 19%, 8%, 2% and 0% at ages 1, 2, 3 and 4 mo, respectively. Complementary foods and family foods were introduced at median ages of 2.5 and 6.3 mo, i.e. much earlier than recommended. Better adherence to recommendations was associated with smaller number of children in the family, increased maternal education and some other socio-economic or environmental variables. CONCLUSION: Exclusive breastfeeding is uncommon and complementary foods were introduced early to newborns among these rural families. Education and family planning may improve adherence to infant feeding recommendations and reduce the incidence of early childhood malnutrition in Malawi.

Gestational health and predictors of newborn weight amongst pregnant women in rural Malawi.

This study was conducted to provide community-based data on maternal health and predictors of newborn weight in rural Malawi. Data were obtained prospectively from a community-based cohort of 581 pregnant women who attended an antenatal clinic and delivered a term, live-born, singleton infant in Lungwena, rural Malawi. Morbidity from infectious diseases and anaemia was common. Maternal weight gain in rural Malawi was slower but fundal height gain was comparable to that of an affluent western population. The mean +/- SD weight of term newborns was 3.2 +/- 0.5 kilograms. A regression model including data from all routine investigations explained only 24% of the variance in newborn weights, suggesting that routine antenatal measurements had a limited power to predict the size of term live-born babies. Maternal parity, initial weight, the duration of pregnancy and gestational weight gain were associated with newborn weights and should, therefore, be systematically recorded in rural Malawian antenatal clinics.

Epidemiology and predictors of infant morbidity in rural Malawi.

In rural Malawi, 703 newborns were visited monthly for 1 year to describe the epidemiology and health-seeking behaviour during acute episodes of diarrhoea, respiratory infections (ARI) and malaria. On average, the infants suffered from 1.3 annual episodes (11.0 illness days) of diarrhoea, 1.1 episodes (9.4 days) of ARI and 0.7 episodes (4.8 days) of malaria. Multivariate analysis with polychotomous logistic regression indicated that the amount of morbidity was associated with the child's area of residence, weight in early life, number of siblings, father's marital status and the source of drinking water. Diarrhoea and malaria were most common at 6-12 months of age and during the rainy months whereas respiratory infections peaked at 1-3 months of age and in the cold season. Ten per cent of diarrhoea, 9% of ARI and 7% of malaria episodes lasted for more than 14 days. Fifty-eight infants died, giving case fatality rates of 1% for diarrhoea, 2% for ARI and 4% for malaria. One-third (37%) of the illness episodes were managed at home without external advice. A traditional healer was consulted in 16% of episodes and a medical professional in 55% of episodes. If consulted, traditional healers were seen earlier than medical professionals (median duration after the onset of symptoms 0.7 vs. 1.8 days, P < 0.001). Traditional healers were significantly more commonly used by those families whose infants died than by those whose infants did not die (odds ratio 1.8, 95% CI 1.1, 3.0). Our results emphasise the influence of seasonality, care and living conditions on the morbidity of infants in rural Malawi. Case fatality for diarrhoea, ARI and malaria was high and associated with health-seeking behaviour among the guardians. Future interventions must aim at early and appropriate management of common childhood illnesses during infancy.

The relationship between antenatal risk characteristics, place of delivery and adverse delivery outcome in rural Malawi.

OBJECTIVE: To describe and compare the frequency of antenatally identified maternal 'risk' characteristics, place of delivery and occurrence of delivery complications. METHODS: A prospective cohort study of 780 pregnant women completing antenatal follow-up at a rural health center in Malawi. RESULTS: Three-quarters of the subjects had at least one commonly accepted risk characteristic. Only 30% of these women, and 22% of those with no risk characteristics, delivered in a modern health facility. Four women died, 127 experienced other delivery complications and there were 52 perinatal deaths. The 'at-risk' classification had over 80% sensitivity but less than 30% specificity to predict delivery complications or perinatal deaths. The positive predictive values were as low as 20% for delivery complications and 7% for perinatal mortality. Most individual 'risk' characteristics were not associated with adverse delivery outcomes, even when adjusted for the place of delivery. CONCLUSIONS: Antenatal risk identification failed to promote safe deliveries because of a poor predictive value of the 'risk' variables and the failure of the identified 'at-risk' individuals to deliver in modern health facilities.

The importance of preterm births for peri- and neonatal mortality in rural Malawi.

Peri- and neonatal mortality remain high in developing countries, especially in sub-Saharan Africa. In the present study, we quantified and identified the most important predictors of early mortality in rural Malawi. Data were obtained from a community-based cohort of 795 pregnant women and their 813 fetuses, followed prospectively from mid-pregnancy. In this group, peri- and neonatal mortality rates were 65.3 deaths per 1000 births and 37.0 deaths per 1000 live births respectively. When controlled for month of birth, maternal age and selected socio-economic variables, preterm birth was the strongest independent predictor of both peri- and neonatal mortality (adjusted odds ratios 9.6 for perinatal and 11.0 for neonatal mortality; 95% confidence intervals: [4.4, 21.0] and [3.7, 32.7] respectively). Weaker risk factors for mortality included a maternal history of stillbirth and abnormal delivery. Preterm delivery was associated with primiparity and peripheral malaria parasitaemia of the mother, and it accounted for 65% of the population-attributable risk for perinatal and 68% of the neonatal mortality. Successful intervention programmes to reduce peri- and neonatal mortality in Malawi have to include strategies to predict and prevent prematurity.

Antenatal and perinatal predictors of infant mortality in rural Malawi.

BACKGROUND: The slow pace in the reduction of infant mortality in sub-Saharan Africa has partially been attributed to the epidemic of human immunodeficiency virus (HIV) infection. To facilitate early interventions, antenatal and perinatal predictors of 1st year mortality were identified in a rural community in southern Malawi. METHODS: A cohort of 733 live born infants was studied prospectively from approximately 24 gestation weeks onwards. Univariate analysis was used to determine relative risks for infant mortality after selected antenatal and perinatal exposures. Multivariate modelling was used to control for potential confounders. FINDINGS: The infant mortality rate was 136 deaths/1000 live births. Among singleton newborns, the strongest antenatal and perinatal predictors of mortality were birth between May and July, maternal primiparity, birth before 38th gestation week, and maternal HIV infection. Theoretically, exposure to these variables accounted for 22%, 22%, 17%, and 15% of the population attributable risk for infant mortality, respectively. INTERPRETATION: The HIV epidemic was an important but not the main determinant of infant mortality. Interventions targetting the offspring of primiparous women or infants born between May and July or prevention of prematurity would all have considerable impact on infant survival.

Effects of heme proteins on nitric oxide levels and cell viability in isolated PMNs: a mechanism of toxicity.

Isolated human PMNs served as a model to determine oxyhemoglobin (oxyHb) binding and the effects of oxymyoglobin (oxyMb) or oxyHb on production of both nitric oxide (NO*) and superoxide (O2*-) and the resulting cytotoxicity. Physiologically relevant concentrations of NO* and H2O2 oxidized, to a similar extent, 2,7-dichlorodihydrofluorescein (DCFH) loaded into polymorphonuclear neutrophils (PMNs). Activation of PMNs with phorbol 12-myristate 13-acetate (PMA) markedly increased the internalization of extracellular oxyHb (10-250 microg/mL). OxyMb (10-300 microg/mL) or oxyHb (30-300 microg/mL) enhanced DCFH oxidation by a concentration-dependent mechanism in unstimulated, lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNF-alpha)-, and PMA-stimulated PMNs. This increased DCFH oxidation was eliminated by NO* synthase inhibitors, glutathione and ascorbate, and was reduced by albumin. Nitrite accumulation in PMN filtrates mirrored NO*-induced DCF fluorescence. OxyMb-induced increases in NO* levels paralleled alterations in DNA and cell membrane damage and ATP levels in PMNs and co-cultured lymphocytes, and were attenuated by NO* synthase inhibitors. OxyMb eliminated extracellular O2*- at protein concentrations 100- to 1000-fold above those of superoxide dismutase. These results suggest that heme proteins bind and internalize into PMNs and increase NO*-induced damage in neighboring cells by inhibiting O2*(-)-scavenging of NO*. We propose a mechanism whereby heme protein-induced NO* levels may contribute to immunosuppression and increased infection rates associated with transfusions and cellular damage during inflammation.

Socio-economic support for good health in rural Malawi.

OBJECTIVE: To study the socio-economic support for good health among subsistence farmers in rural Malawi. DESIGN: A cross-sectional survey. SETTING: Lungwena, a rural area with 17,000 inhabitants in southern Malawi. PARTICIPANTS: Seven hundred and ninety five pregnant women who attended the antenatal clinic at Lungwena Health Centre between June 1995 and September 1996. INTERVENTIONS: Interviews about socio-economic conditions. Measurements of cultivated land areas and distances between home and the local health centre. MAIN OUTCOME MEASURES: Proportion of households lacking literate adults, adequate water source and sanitation, easy access to modern health care or food security. RESULTS: Only 14% of the interviewed women could read and write and half of the households had no literate members. Every fifth household was lacking both an access to safe drinking water and a proper sanitary facility. The distance to the health centre was more than 5 km among half of the households and only 37% had enough land to grow food for all family members. When other potential means of obtaining food were taken into account, 27% of the households had no food security. Numerous households were lacking more than one socio-economic prerequisites of good health: three or more were missing from a quarter of the families. CONCLUSIONS: Socio-economic prerequisites of health were commonly missing in Lungwena. Subsequent health interventions should strengthen the investments into general poverty alleviation.

Archaeal promoter-directed expression of the Halobacterium salinarum catalase-peroxidase gene.

The Halobacterium salinarum catalase-peroxidase gene was subcloned into shuttle vectors pWL102 and pWL202 and expressed under the control of different archaeal promoters. When Hbt. salinarum was transformed with the catalase-peroxidase gene under the control of its own promoter, catalase-peroxidase activity increased twofold. Catalase-peroxidase activity increased threefold when Hbt. salinarum was transformed with the catalase-peroxidase gene under the control of a tRNA promoter. This bifunctional enzyme in Hbt. salinarum was not induced by environmental stresses such as H2O2, intense light, darkness, high temperature, low temperature, redox inhibitors, heavy metals, or ions.

Childhood immunization in rural Malawi: time of administration and predictors of non-compliance.

A cohort of 760 newborns was followed prospectively for 2 years to ascertain the time of administration of childhood vaccinations in rural Malawi and to study predictors of non-compliance with national vaccination recommendations. At 1 year of age, 99% of the infants were fully vaccinated against tuberculosis, 91% against polio, 90% against diphtheria, pertussis and tetanus and 64% against measles. At 2 years, the corresponding vaccination coverages were 99%, 93%, 93% and 84%. On average, all vaccinations were given 1-3 months later than recommended. Many of the delayed measles vaccinations were given during a separate vertical campaign, during which 25% of previously unvaccinated 21-23-month-old children were identified and immunized. Non-compliance with vaccination recommendations was associated with living in villages with no access to mobile vaccination teams, birth between April and June and birth at home. In this rural Malawian area, most vaccination services were functioning well. To increase measles vaccination coverage, regular outreach activities should be encouraged.

Photooxidative bleaching of chlorophyllin.

Chlorophyllin, a water-soluble, copper-containing porphyrin, can be bleached rapidly in the light or slowly in the dark in a reaction which is oxygen dependent. Both the photo and the dark bleaching reactions are temperature dependent. Cyclic voltammetry measurements show that the copper in the bleached and nonbleached state remains in the +2 redox state and could be readily reduced. This would imply that there is no net oxidative change to the copper during the bleaching process. FT-IR absorption spectroscopy showed vibrations characteristic of a vinyl functionality disappeared upon bleaching. Aqueous solutions of chlorophyllin were not dialyzable through dialysis tubing of molecular weight cut-off, 6000-8000 molecular weight, indicative of an aggregate chlorophyllin micelle. Analysis of products by high performance liquid chromatography showed that the chlorophyllin mixture was more complex than originally anticipated and that two components were lost from the mixture upon photobleaching. One compound that is preferentially lost upon photobleaching has been identified by mass spectral analysis as Cu(II) chlorin e6.

Active oxygen intermediates and chlorophyllin bleaching.

Bleaching of chlorophyllin, a water soluble copper containing porphyrin molecule, was investigated with regard to the potential role of active oxygen intermediate involvement. It was found that the bleaching was highly aerobic and also biphasic in nature. The aerobic photobleaching and the dark bleaching were effectively prevented by the addition of reductants such as ascorbate and cysteine. In addition, the reductant and peroxyl radical scavenger, Trolox, was highly effective in preventing bleaching. Catalase was moderately effective in preventing photobleaching whereas peroxidase and superoxide dismutase hastened the photobleaching process. It is concluded that the bleaching of chlorophyllin is a peroxidative process which does not involve singlet oxygen, superoxide, nor the .OH radical.

The iron-containing superoxide dismutase-encoding gene from Chlamydomonas reinhardtii obtained by direct and inverse PCR.

The complete sequence of the Fe2+ -containing superoxide dismutase (Fe-SOD)-encoding gene of Chlamydomonas reinhardtii was determined from genomic DNA by use of the direct (PCR) and inverse PCR (IPCR). It was confirmed by reverse transcription-PCR. Primers employed in the PCR represented oligodeoxyribonucleotides corresponding to conserved amino acid (aa) residues of Fe-SOD. From the sequence of direct PCR product, primers were designed for IPCR. All amplified fragments were cloned and sequenced. Analysis of Fe-SOD reveals that the open reading frame consists of 234 aa which show a high degree of homology to other Fe-SOD. The first 33 aa are predominantly either hydrophobic or basic, and may serve as the signal peptide for this chloroplastic protein. Potential transcription start points and eukaryotic control elements, as well as a polyadenylation site, were identified.

Alterations in oxidative activity and superoxide dismutase in Halobacterium halobium in response to aerobic respiratory inhibitors.

Aerobic incubation of Halobacterium halobium with low concentrations of cyanide or azide resulted in a twofold increase in activity of the Mn-containing superoxide dismutase (SOD). A similar effect was observed with antimycin A, albeit at a higher concentration. Oxygen consumption increased 100% at concentrations of cyanide, azide, and antimycin A that produced maximal SOD induction. Luminol chemiluminescence of H. halobium cells, indicative of oxidative activity, was elevated in the presence of concentrations of aerobic respiratory inhibitors that corresponded to maximal levels of SOD induction. In addition, oxidation of NADH by cell extracts was maximal at inhibitor concentrations that correlated with the peak induction of SOD. Proton fluxes for H. halobium cells also corresponded to concentration ranges of inhibitor resulting in greatest SOD levels, indicative of a potential uncoupling effect. A similar phenomenon was also observed with the uncoupler carbonyl cyanide m-chlorophenyl hydrazone. Growth was unaffected by inhibitors at the concentrations used for SOD induction. The results are interpreted as an induction of enhanced levels of SOD through a combination of increased electron flow and an increased oxidative environment.

Purification and characterization of a mesohalic catalase from the halophilic bacterium Halobacterium halobium.

When subjected to the stress of growth in a relatively low-salt environment (1.25 M NaCl), the halophilic bacterium Halobacterium halobium induces a catalase. The protein has been purified to electrophoretic homogeneity and has an M(r) of 240,000 and a subunit size of approximately 62,000. The enzyme is active over a broad pH range of 6.5 to 10.0, with a peak in activity at pH 7.0. It has an isoelectric point of 4.0. This catalse, which is not readily reduced by dithionite, shows a Soret peak at 406 nm. Cyanide and azide inhibit the enzyme at micromolar concentrations, whereas maleimide is without effect. The addition of 20 mM 3-amino-1,2,4-triazole results in a 33% inhibition in enzymatic activity. The tetrameric protein binds NADP in a 1:1 ratio but does not peroxidize NADPH, NADH, or ascorbate. Although the enzymatic activity is maximal when assayed in a 50 mM potassium phosphate buffer with no NaCl, prolonged incubation in a buffer lacking NaCl results in inactive enzyme. Moreover, purification must be performed in the presence of 2 M NaCl. Equally as effective in retaining enzymatic function are NaCl, LiCl, KCl, CsCl, and NH4Cl, whereas divalent salts such as MgCl2 and CaCl2 result in the immediate loss of activity. The catalase is stained by pararosaniline, which is indicative of a glycosidic linkage. The Km for H2O2 is 60 mM, with inhibition observed at concentrations in excess of 90 mM. Thus, the mesohalic catalase purified from H. halobium seems to be similar to other catalases, except for the salt requirements, but differs markedly from the constitutive halobacterial hydroperoxidase.

Enhanced superoxide production by membrane vesicles from Halobacterium halobium in a hyposaline environment.

Membrane vesicles were prepared from the halophilic archaebacterium, Halobacterium halobium, which was grown either in medium containing 4 M NaCl or in a relatively hyposaline medium containing 1.25 M NaCl. Membrane vesicles prepared from bacteria grown in the lower salt environment consumed more oxygen, oxidized more NADH and generated more superoxide than vesicles prepared from cells grown in the normal 4 M NaCl containing medium. The enhanced respiratory activity of the membrane fragments obtained from the halophile which was grown and assayed in a hyposaline environment, along with the concomitant increased flux in superoxide, demonstrate a relation between an environmental perturbation and an altered electron transport activity.

Purification of a catalase-peroxidase from Halobacterium halobium: characterization of some unique properties of the halophilic enzyme.

A hydroperoxidase purified from the halophilic archaeon Halobacterium halobium exhibited both catalase and peroxidase activities, which were greatly diminished in a low-salt environment. Therefore, the purification was carried out in 2 M NaCl. Purified protein exhibited catalase activity over the narrow pH range of 6.0 to 7.5 and exhibited peroxidase activity between pH 6.5 and 8.0. Peroxidase activity was maximal at NaCl concentrations above 1 M, although catalase activity required 2 M NaCl for optimal function. Catalase activity was greatest at 50 degrees C; at 90 degrees C, the enzymatic activity was 20% greater than at 25 degrees C. Peroxidase activity decreased rapidly above its maximum at 40 degrees C. An activation energy of 2.5 kcal (ca. 10 kJ)/mol was calculated for catalase, and an activation energy of 4.0 kcal (ca. 17 kJ)/mol was calculated for peroxidase. Catalase activity was not inhibited by 3-amino-1,2,4-triazole but was inhibited by KCN and NaN3 (apparent Ki [KiApp] of 50 and 67.5 microM, respectively). Peroxidative activity was inhibited equally by KCN and NaN3 (KiApp for both, approximately 30 microM). The absorption spectrum showed a Soret peak at 404 nm, and there was no apparent reduction by dithionite. A heme content of 1.43 per tetramer was determined. The protein has a pI of 3.8 and an M(r) of 240,000 and consists of four subunits of 60,300 each.

Elevation of superoxide dismutase in Halobacterium halobium by heat shock.

Exposure of Halobacterium halobium to 50 degrees C for 2.5 h in an aerobic environment resulted in a greater than twofold increase in the activity of the manganese-containing superoxide dismutase. Nondenaturing polyacrylamide gels stained for enzymatic activity did not reveal any additional isozymes of superoxide dismutase induced by the heat shock. The maximal effect was observed at 50 degrees C, and the elevated levels of activity remained constant during 5 h of recovery at 40 degrees C. The induction of enzymatic activity was sensitive to protein synthesis inhibitors. The results are discussed relative to heat shock and stress-related proteins as well as alterations in metabolism brought about by elevated temperatures.